For many years we have been studying how these polyamines are synthesized, how their biosynthesis and degradation are regulated, their physiologic functions, how they act in vivo, and the structure of the various biosynthetic enzymes. For this purpose we have constructed null mutants in each of the biosynthetic steps in both Escherichia coli and Saccharomyces cerevisiae, and have prepared over-expression systems for the biosynthetic enzymes. Our overall studies have aimed at the use of these mutants to elucidate the physiological functions of the polyamines. Last year we a reported the construction of a strain of Escherichia coli that contained deletions in all of the genes involved in polyamine biosynthesis;namely, speA (arginine decarboxylase), speB (agmatine ureohydrolase), speC (ornithine decarboxylase), spe D (adenosylmethionine decarboxylase), speE (spermidine synthase), speF (inducible ornithine decarboxylase), cadA (lysine decarboxylase), and ldcC (lysine decarboxylase). Despite the complete absence of all of the polyamines, the strain grew indefinitely in air in amine-free media;albeit at a slightly (ca 40-50%) reduced growth rate. Recently we made the unexpected finding that addition of another mutation (lysA) resulted in a strain that had an almost absolute requirement for spermidine for growth. Since most of these mutations had been obtained over many years by treatment of the strains with rather non-specific mutagenic agents, we are now constructing a new strain that only contains mutations specifically located in the desired genes in a well-defined background. . Our present studies in E. coli have been particularly concerned with glutathionylspermidine. As we have previously shown, all of the spermidine of the E. coli cells and a large percentage of the intracellular glutathione are converted to glutathionylspermidine at the end of the logarithmic growth. In our current studies we have developed chromatographic methods for the assay of glutathionylspermidine, and, in contrast to our earlier work, have concentrated on the formation of glutathionylspermidine in cultures still growing logarithmically. The development of these assays has been facilitated by using as a control a strain that does not contain the gene (&#916;gsp) for glutathionylspermdine biosynthesis. We have also constructed mutants of the above strains that require arginine and lysine for growth in order to facilitate proteomic studies by the SILAC technique.